Creatinine iminohydrolase is an enzyme which specifically hydrolyzes creatinine to ammonia. Accordingly, by contacting an aqueous liquid containing creatinine with this enzyme to generate ammonia, the presence and/or concentration of creatinine in the liquid can be determined by detecting the level of generated ammonia. This enzyme can therefore play an important role in the clinical laboratory where it can be used as a diagnostic test reagent for the determination of creatinine in biological liquids.
Creatinine iminohydrolase, sometimes referred to as creatinine desimidase, has been obtained from various microorganisms. For example, J. Szulmajster in J. Bacteriolol, 75: 633 (1958) and Biochim Biophys Acta, 30: 154 (1958) describes a preparation of creatinine iminohydrolase obtained from the anaerobic, gram-positive microorganism Clostridium parapurtrificum. A method of growing the Clostridium parapurtrificum microorganism is also described in these Szulmajster publications. However, these publications relate specifically to an anaerobic, gram-positive microorganism, and the disclosed fermentation method for growing the microorganism requires a long time. Moreover, the amount of microbial cells grown and the yield of enzyme extracted therefrom is relatively small.
U.S. Pat. Nos. 4,087,329 and 4,134,793 describe the production of the enzyme creatinine desimidase from one of several aerobic microbial sources including microorganisms of the genera Brevibacterium, Corynebacterium, Pseudomonas, and Arthrobacter. These patents further describe a nutrient medium which may be used for culturing microorganism of the aforementioned genera. These patents assert that the formulation of this nutrient medium can be widely varied and can contain any of a large number of specifically recited carbon and nitrogen sources, as well as other optional nutrients, including inorganic materials, a creatinine inducer, and the like.
Goodhue, Esders, and Masurekar U.S. Pat. No. 4,276,377 describes and claims a creatinine iminohydrolase enzyme preparation free from urease activity obtained from an aerobic soil microorganism, preferably the aerobic soil microorganism ATCC 31546. Because this enzyme preparation is free of urease contamination and is highly specific for creatinine, creatinine assays can be performed with this enzyme without regard to interference by urea and other nitrogenous substances that are often present in biological aqueous liquids to be assayed for creatinine, e.g., serum. The urease-free creatinine iminohydrolase enzyme preparation described therein is therefore highly desirable.
Masurekar's U.S. Pat. No. 4,275,164 describes a process for the production of creatinine iminohydrolase from an aerobic soil microorganism by transferring a growth microorganism to a production medium having a pH in the range 5 to 10 and extracting urease-free creatinine iminohydrolase. The production medium comprises a carbon source containing glucose or an amino acid precursor, a nitrogen source containing creatinine, trace nutrients and a buffer. Although the process produces excellent enzyme activity (as units per liter) and high specific activity (as units per gram dry cell mass), further improvement is susceptible to the toxic effects of fermentation medium components at high concentration and metabolites such as N-methylhydantoin which decrease productivity and yield. In addition, the use of complex nutrient sources such as yeast extract makes the fermentation susceptible to contamination by foreign microbes which decrease the fermentation productivity and enzyme activity, as well as themselves being a source of impurities that make subsequent enzyme recovery and purification more difficult.
An improved process from that described in U.S. Pat. No. 4,275,164 for providing a creatinine iminohydrolase with increased yield would represent a clearly advantageous addition to the art. Such a process and nutrient medium used therein would be particularly desirable if useful with the aerobic soil microorganism ATCC 31546.